We continue to focus our efforts to elucidate: i) the catalytic cycle and transport pathway of human P-gp; ii) the molecular basis of the polyspecificity of P-gp; iii) the interaction of clinically relevant tyrosine kinase inhibitors (TKIs) and other natural product modulators with P-gp and ABCG2; iv) pharmacophore features required for binding of third generation cyclic peptide inhibitor derivatives to P-gp and ABCG2; v) the use of single particle cryo-EM for the analysis of the conformational landscape of human P-gp during its catalytic cycle and vi) identification of the epitopes for three human P-gp-specific monoclonal antibodies (MRK-16, UIC2 and 4E3). We have been employing cell-based, biochemical, biophysical, pharmacological, and physiological techniques along with molecular biology and molecular modeling approaches to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we have devoted considerable effort to the screening and development of TKIs and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp and the role of conserved motifs in the ATP-binding cassette: We are continuing our studies on the catalytic cycle and transport pathway of P-gp. We are using molecular modeling and mutagenesis approaches to elucidate on a molecular level how this transporter recognizes and transports a wide variety of structurally dissimilar compounds. One goal is to understand the role of TMH1 and TMH7 in binding and transport of substrates. TMH1 and TMH7 are topologically identical helices in the two halves of P-gp and may together regulate substrate transport. We selected six conserved residues each from TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732) that appear to be part of the drug-binding pocket. These residues were substituted with alanine by gene synthesis to generate TMH1,7 mutant P-gp. We found that TMH1,7 mutant P-gp with twelve substitutions was expressed normally in HeLa cells, but was only able to transport three of the 25 tested substrates. We also observed partial loss of ATPase activity and lack of stimulation by the substrates. Based on these results, we propose that TMH1 and TMH7 play a critical role in the translocation step of the transport cycle of P-gp. 2. The mechanism of the molecular basis of polyspecificity, which is an important property of multidrug transporters, by using molecular modeling and mutagenesis approaches: Towards the goal of understanding the molecular basis of the broad substrate specificity of P-gp, we characterized the effect of a detergent micelle environment on the drug-mediated inhibition of P-gp ATPase activity. Most of the substrates or modulators of P-gp stimulate its basal ATPase activity, and only a few drugs have been found to inhibit it. Last year we demonstrated that the high-affinity drug-binding site is inaccessible either due to a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. Thus, the structural and functional aspects of ligand interactions with purified P-gp need to be studied in a detergent-free native or artificial membrane environment. We have optimized the conditions to reconstitute purified protein in nanodiscs. We found that inclusion of 0.1% cholesteryl hemisuccinate during purification and preparation of nanodiscs using bacterial polar lipid mixture and MSP1 belt protein stabilizes the P-gp. The interaction of substrates and modulators with purified P-gp in nanodiscs is similar to that observed in native membrane vesicles. Currently, we are using P-gp reconstituted in nanodiscs to determine the affinities of substrates with surface plasmon resonance and cellular thermal shift assays. 3. Resolution of the three-dimensional structure of human Pgp: The resolution of the three-dimensional structure of P-gp is an ongoing project and for this we have developed a purification scheme that has yielded total protein of 7.5-10.0 mg of homogeneously pure P-gp. Due to the flexible nature of human P-gp, we are also using single particle analysis by the cryo-electron microscopy technique. To improve the resolution to an atomic level, we are using purified human P-gp reconstituted in nanodiscs. We have optimized the conditions for reconstituting purified P-gp in nanodiscs in a functional form. The EM analysis of negatively stained P-gp-nanodisc samples in the absence and presence of ATP-Mg, ADP-vanadate and Fab of UIC2 antibody suggests that there is a single molecule of P-gp per nanodisc. We are currently using human P-gp reconstituted in nanodiscs to obtain the atomic level structure of the closed (ADP-vanadate trapped post hydrolysis) conformation by cryo-EM. The single particle cryo-EM studies were carried out in collaboration with Dr. Sriram Subramaniam. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We have extended these studies by synthesizing over 100 derivatives of the cyclic peptide inhibitor TTT28 in collaboration with Dr. Tanaji Talele (St. John's University, NY). We found that at least two derivatives inhibited the ATPase activity of P-gp and sensitized cells expressing P-gp and ABCB2 to anticancer drugs. Docking studies indicated that these derivatives bind to the drug-binding pocket of P-gp. To validate the docking poses of one of the inhibitory derivatives (compound 109), we used single or double mutations in the drug-binding pocket. In addition, we continue to characterize recently developed tyrosine kinase inhibitors, which are used in the clinic to treat cancer patients, for their effect on the function of P-gp and ABCG2. The orally available selective inhibitor of histone deacetylase 6 histone, ricolinostat appears to be a substrate for both P-gp and ABCG2. On the other hand, SIS3, a specific inhibitor of Smad3 modulates the function of P-gp and ABCG2. In addition, we found that the positive inotropic agent DPI-201106 inhibited the transport function of P-gp. This agent also partially inhibited ATP hydrolysis by P-gp, suggesting that either it interacts with P-gp as a high-affinity substrate similar to cyclosporine A or as a modulator. These studies were carried out in collaboration with Dr. Chung-Pu Wu (Chang Gung University, Taiwan). 5. Identification of epitopes for three human P-gp-specific monoclonal antibodies (MRK-16, UIC2 and 4E3). Antibody-mediated inhibition of human P-gp activity has been shown to overcome resistance to anticancer drugs. However, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. To identify the epitopes, we generated a series of human-mouse P-gp chimeras. By replacing specific mouse amino acids in the ECL1 and ECL4 with human P-gp residues, we determined the amino acids required for the binding of the three antibodies. By substituting just thirteen residues in ECL1 and five in ECL4 in mouse P-gp with human amino acids, we demonstrate recovery of the conformation-sensitive binding of UIC2 to mouse P-gp. The in-silico docking of the antigen-binding fragment (Fab) region of MRK-16 and UIC2 to a homology model of human P-gp supported the experimental results. Thus, we demonstrate that the discontinuous epitopes for three human P-gp-specific monoclonal antibodies are overlapping and located in ECLs 1 and 4 of human P-gp. These findings will help to devise immunotherapy to target P-gp to overcome resistance to chemotherapy in cancer patients.